Nucleic acids encoding (poly) peptides having chips activity

ABSTRACT

The invention relates to nucleic acid molecules encoding (poly)peptides having CHIPS activity, to recombinant vectors harboring such molecules, and the host cells carrying the vectors. The invention further relates to methods for preparing recombinant (poly)peptides having CHIPS activity and to the use of such recombinant (poly)peptides having CHIPS activity for diagnosis, prophylaxis and treatment, such as the treatment of inflammation reactions and HIV. In addition the invention provides therapeutic and diagnostic compositions comprising as the active ingredient the (poly)peptide having CHIPS activity.

[0001] The present invention relates to a nucleic acid molecule encodinga (poly)peptide having CHIPS activity. The invention further relates tothe use of the information contained in the nucleic acid for thepreparation of the corresponding (poly)peptide and to vectors and hostsfor use therein. The invention in addition relates to non-(poly)peptidemolecules having a similar structure and function as the (poly)peptides.The (poly)peptide having CHIPS activity that is encoded by the nucleicacid molecule of the invention can be used in the treatment ofinflammation reactions. The (poly)peptides and non-(poly)peptides can inaddition be used for inhibiting activation of leukocytes and endothelialcells.

[0002] Leukocytes are mainly involved in protecting the body againstforeign invaders (e.g. bacteria, viruses, fungi, and cancer cells). Themost important cells are lymphocytes, monocytes and neutrophils.Lymphocytes form the specific immune system and cause immune reactionsagainst invaders. Their most important task is to build up specificmemory against the invader, so that the next time the invader enters thebody it is recognised, killed and removed rapidly. Sometimes theselymphocytes not only attack invaders, but also react against certainstructures and/or molecules (so-called auto-antigens) of the own body,causing auto-immune diseases (e.g. rheumatoid arthritis). Killing andremoval of invaders is mostly done by monocytes and neutrophils, cellsof the innate immune system, by direct recognition of the invaders orwith the help of specific lymphocytes.

[0003] In contrast to the delicate network of the fine-tuned andcontrolled reactions of lymphocytes, cells of the innate system react ina relatively non-specific and aggressive way. Since they are part of thebody's first line of defence, their most important task is to kill andremove the invading agent as quickly as possible. This is accomplishedthrough very aggressive substances (e.g. free radicals and enzymes) thatare not only lethal to the invader, but also cause damage to host cellsin the vicinity. Substances from these damaged cells and the locallyactivated cells from the innate system itself will further attractincreasing numbers of neutrophils and monocytes, causing localinflammation. In most cases, such an aggressive and damaginginflammatory reaction, caused by overactivated neutrophils, isunnecessary. In some cases this inflammatory response is responsible forserious, sometimes lethal disorders and includes conditions like AdultRespiratory Distress Syndrome (ARDS), severe tissue damage followingthrombotic events such as heart attacks and stroke, inflammatory boweldiseases and rheumatoid arthritis. The inflammation will subside onceall the invaders have been killed and removed, together with the variouscells killed in the process. Healing of the wound, caused by theinflammatory response, can then begin. Although there is some overlap infunction, the main task of neutrophils is to attack the invaders and themain task of monocytes is to remove the debris resulting from thisattack. In addition, neutrophils have another peaceful task in assistingthe wound healing process.

[0004] When bacteria have invaded the body and, for example, infectedthe central nervous system (as in meningitis) they start to producemicrobial substances, including the formulated polypeptides (like thefMLP peptide). Other substances of microbial origin activate thecomplement factor 5 (CS) convertase enzyme-complex, that converts C5 ofthe complement system into its activated C5a form. Both C5a and fMLP arechemo-attractants: substances that can activate and attract cells fromthe blood vessels (the migration process). Neutrophils are responsive tothese two substances and also to interleukin-8 (IL-8). This “chemokine”(the name given to chemo-attractants that are produced by cells of theimmune system) is produced mainly by activated monocytes (but also inminute amounts by the activated neutrophils themselves). Neutrophilsinteract with these substances, because they have receptors for thesesubstances on the outside of their cell membrane.

[0005] Activated neutrophils can easily migrate from blood vessels. Thisis because the chemo-attractants, microbial products and substances fromactivated monocytes will have increased the permeability of the vesselsand stimulated the endothelial cells of the vessel walls to expresscertain adhesion molecules. Neutrophils express selectins and integrins(e.g. CD11/CD18) that bind to these adhesion molecules. Once theneutrophil has adhered to the endothelial cells, it is able to migratethrough the cells, under the guidance of chemo-attractants/chemokines,towards the site of infection, where the concentration of thesesubstances is at its highest. These substances also activate neutrophilsto produce a range of other molecules, some of which attract moreneutrophils (and subsequently monocytes), but, mostly, they areresponsible for destroying the invading bacteria. Some of thesesubstances (e.g. free radicals, enzymes that break down proteins(proteases) and cell membranes (lipases)) are so reactive andnon-specific that cells from the surrounding tissue (and the neutrophilsthemselves) are destroyed, causing tissue damage. This damage isexacerbated by the presence of blood derived fluid which hastransgressed the leaky vessel wall and is responsible for the swellingthat always accompanies inflammation (called oedema). The pressure buildup caused by this excess fluid results in further cell damage and death.

[0006] Later in the inflammatory process, monocytes migrate to the sceneand become activated. Besides their role in removing bacteria and celldebris, they also produce substances such as tumour necrosis factor(TNF) and IL-8, which in turn attract more activated neutrophils,causing further local damage. TNF also has a direct stimulatory effecton neutrophils. Once all the invaders have been removed, theinflammatory response will subside and the area will be cleared of theremaining ‘casualties’. Then the process of wound healing starts.Although it is known that neutrophils play a pivotal role in woundhealing, it is not clear which neutrophil-derived substances areinvolved and how the neutrophils are active in healing without beingaggressive to the surrounding tissue. In general, damaged tissue will bereplaced by scar tissue formed mainly of fibroblasts and collagen. Wheninflammation occurs in areas of the body with an important function,like tissues formed from heart muscle cells, brain cells or lungalveolar cells, normal function will be compromised by the resultingscar formation, causing serious conditions like heart failure, paralysisand emphysema. To minimise the debilitating consequences of theseconditions, it is important to ‘dampen’ the inflammatory reaction asquickly as possible.

[0007] Intervention to control the acute early phase inflammatoryresponse presents an opportunity to improve the prognosis for a widerange of patients whose symptoms can be traced back to such an event.Such an approach has been advocated for many acute and chronicinflammation-based diseases and shown to have potential based onfindings from relevant disease models. Classical anti-inflammatory drugssuch as steroids and Non Steroid Anti-Inflammatory Drugs (NSAIDS) do nothave the ideal profile of action, either in terms of efficacy or safety.Steroids affect the ‘wrong’ cell type (monocytes) and their dampeningeffects are easily bypassed. NSAIDS generally show a relatively mildeffect partly because they intervene at a late stage in the inflammatoryprocess. Both classes of drugs produce a range of undesirable sideeffects resulting from other aspects of their pharmacological activity.Drugs acting directly and specifically to prevent migration andactivation of neutrophils may have a number of advantages. Several drugsunder early development only interfere with one individual aspect ofneutrophil activation (e.g. C5 convertase inhibitors, antibodies againstC5a, C5a-receptor blocking drugs) and migration (antibodies againstintegrins (like. CD11b/CD18) and L-selectin on neutrophils andantibodies against adhesion molecules (like ICAM-1 and E-selectin) onendothelial cells). Antibodies against TNF and IL-8 have effects inchronic inflammation, but only marginal effects in acute inflammation,because of the minimal role monocytes (which are mainly responsible forthese substances' production) play in the acute phase.

[0008] Sometimes, the cause of the acute inflammation cannot be removedand the inflammation becomes chronic. With the exception oftuberculosis, chronic hepatitis and certain other conditions, this isseldom the case with infections. However, chronic inflammation can alsobe caused by stimuli other than bacteria, such as auto-immune reactions.Research has shown that in chronic inflammation the role of monocytes ismuch more prominent, and that neutrophil migration and activation,monocyte migration and activation, tissue damage, removal of dead cellsand wound healing are all going on at the same time. This complexcascade of interactions between cells and many different cytokines andchemokines has been the subject of intensive research for many years. Itwas believed that monocytes- and their products were the most importantelements that needed to be inhibited to dampen chronic inflammation.This explains why steroids, which specifically interact with monocytes,are generally more effective in chronic as opposed to acuteinflammation. Long-term treatment with steroids however, is not adesirable option, because severe and unacceptable side effects can occurat the doses required to produce a clinical effect. Newer treatmentsusing antibodies against TNF or IL-8 have shown good results, and wereinitially seen as proof of the major role monocytes were thought to playin chronic inflammation. Recent research casts doubts on an exclusiverole for monocytes in inflammation and points to a critical role forneutrophils, which are now seen to represent better targets fortherapeutic intervention.

[0009] The underlying cause of a chronic inflammatory condition is notalways clear, and the original cause may not always be responsible forfuture recurrence. Some scientists believe that in certain chronicinflammatory diseases there is a continuous cycle of events. Their ideais that existing activated neutrophils and monocytes continuouslyattract and activate new groups of cells, thus perpetuating theinflammatory response even when the initial stimulus is no longerpresent. This would suggest that an acute or periodic treatment with aneffective inhibitor of the neutrophil and monocyte activation would stopthe cycle of new cell recruitment, leading in due course to modificationof disease progression, or even a complete cure, and not justsymptomatic relief.

[0010] In the research that led to the present invention a new agentwith inflammation-inhibiting properties was found in the extracellularmedium of growing Staphylococcus aureus (S. aureus). This agent is thesubject of co-pending application PCT/NL99/00442. The agent was found tobe capable of directly or indirectly blocking different chemokinereceptors. Incubation of different cells with the medium resulted in agreatly reduced expression of a number of the chemokine receptors, bothof the expression of receptors of classical chemotactic agents such asfMLP and C5a on granulocytes and of the expression of CXCR4 and CCR5receptors on lymphocytes, monocytes and macrophages. The reducedreceptor expression was related to greatly reduced chemotaxis relativeto the chemokines, as well as a reduced infection with HIV.

[0011] The activity of the protein is already manifest in the culturesupernatant of the growing S. aureus. The active protein could befurther purified, for example by means of a number of Ligand Dyecolumns. A pre-purification was first performed on a so-called “yellowcolumn” (“Reactive Yellow 86” ligand dye cross-linked 4% beaded agarosecolumn (Sigma)), followed by an absorption chromatography column (theso-called “green column” (“Reactive Green 19” ligand dye cross-linked 4%beaded agarose column (Sigma)) and a DNA column (DNA Cellulose(Pharmacia)). Both latter columns can be interchanged. The DNA columnremoves a contaminant with the same molecular weight as the protein. Theabsorption chromatography column concentrates the protein and isselective for the protein. Finally, a post-purification also takes placeby means of gel filtration and anion exchange chromatography (MonoQ,Pharmacia). In the gel filtration the protein with the molecular weightof about 17 kDa is selected. This is the protein that was found to havechemotaxis inhibitory properties. Because this protein is isolated fromthe supernatant of the Staphylococcus aureus and gives inhibition ofchemotaxis, this protein was named “CHIPS”: CHemotaxis InhibitoryProtein from Staphylococcus aureus (herein also referred to as the“original CHIPS”)

[0012] Isolation of the CHIPS protein out of the supernatant of S.aureus is not very cost-effective. In addition, it is desirable for thepractical use of CHIPS in therapy that the active part of the protein isisolated. Smaller protein or peptide molecules have a reduced risk ofinducing an immunological response in a subject receiving the protein orpeptide for therapy. Furthermore, it is desirable to be able to modifythe protein or peptide to further increase the biological activityand/or lower the immunogenicity thereof.

[0013] It is therefore the object of the present invention to providethe means for producing the original CHIPS protein or othercorresponding (poly)peptides that have CHIPS activity, as well asfunctional fragments, derivatives or analogues thereof other than byisolation from the natural producing host cell.

[0014] The present invention therefore provides a nucleic acid moleculecomprising a nucleotide sequence encoding a (poly)peptide having CHIPSactivity, said nucleotide sequence corresponding to a sequence beingselected from the group consisting of:

[0015] a) a nucleotide sequence comprising at least part of the sequenceas depicted in FIG. 4 (SEQ ID NO 4);

[0016] b) nucleotide sequences encoding a (poly)peptide having CHIPSactivity and having the amino acid sequence depicted in FIG. 5 (SEQ IDNO 5);

[0017] c) nucleotide sequences encoding a (poly)peptide having CHIPSactivity and having at least one portion of the amino acid sequencedepicted in FIG. 5 (SEQ ID NO 5);

[0018] d) nucleotide sequences being at least 40% identical to any oneof the nucleotide sequences a), b) or c);

[0019] e) nucleotide sequences hybridizing at stringent conditions withany one of the nucleotide sequences a), b), c) or d), and

[0020] f) nucleotide sequences complementary to any of the nucleotidesequences a), b), c), d) or e).

[0021] Regarding the identicity or homology as mentioned under d) itshould be noted that for gapped alignments, statistical parameters canbe estimated using the Smith-Waterman algorithm that produces optimalalignment scores. Homologues of the CHIPS nucleic acid sequence orprotein sequence are defined by a Gap Open Penalty of at least 12 and aGap Expression Penalty of at least 1.

[0022] “CHIPS activity” is herein defined as the ability to specificallyimpair at least the responses induced by both fMLP and C5a, including atleast impairment of ligand-(C5a or fMLP) binding, and optionallyimpairment of chemotaxis and cell-activation (e.g. calciummobilization). However, the (poly)peptides may in addition have otherbiological activities, such as an inhibitory effect on the activation ofleukocytes and endothelial cells.

[0023] In the description that follows the terms “CHIPS protein” and“CHIPS gene” or “chp gene” are used for the protein isolated from thesupernatant of naturally occurring S. aureus, and its isolated gene,respectively. “(Poly)peptide having CHIPS activity” and “nucleic acidmolecule encoding a (poly)peptide having CHIPS activity” are used forall other corresponding (poly)peptides and nucleic acid molecules thatare in some way related to or derived from the CHIPS protein or gene buthave an amino acid or nucleotide sequence that is not identical thereto.The CHIPS activity as defined above is an inherent feature of thepresent (poly)peptides. This effect will been demonstrated for the CHIPSprotein in Example 5.

[0024] The sequence as given in FIG. 4 (SEQ ID NO 4) is the DNA sequenceas isolated according to the invention. It comprises a promoter regionfrom nucleotides 1 to 40, a leader peptide sequence from nucleotides 41to 124, the coding region for the (poly)peptide having CHIPSactivity.from nucleotide 125 to 490, as well as a 3′ untranslated regionfrom nucleotides 491 to 603.

[0025] In a first embodiment of the invention, the isolated nucleic acidmolecule has a nucleotide sequence which corresponds to nucleotides 1 to490 of FIG. 4. In an alternative embodiment the promoter region is nolonger present. In this embodiment the nucleotide sequence of thenucleic acid molecule corresponds to nucleotides 41 to 490 of FIG. 4.With this nucleic acid molecule a different promoter and/or othertranscription regulatory sequences can be used. The choice of a promoterand/or other regulatory sequences depends on the conditions under whichtranscription is to take place. The skilled person is capable ofselecting suitable promoter and/or other transcription regulatoryregions.

[0026] The isolated CHIPS gene of FIG. 4 or any nucleic acid derivedtherefrom may for example be operably.linked to the trc expressionsystem (Brosius et al., Gene 27: 161-172 (1984)). Many other suitableexpression control sequences and methods of expressing recombinantproteins are known (F. M. Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Inc., New York, N.Y.).

[0027] The nucleotide sequence as given in FIG. 4 also contains a leaderpeptide sequence. The coding region of the mature protein corresponds tonucleotides 125 to 490 of FIG. 4. Other leader sequences can be used. Orthe leader sequence may be omitted entirely, depending on the host cellin which the sequence is to be expressed.

[0028] The amino acid sequence in FIG. 5 is deduced from the DNAsequence in FIG. 4. In a further embodiment of the invention the nucleicacid molecule thus may have a nucleotide sequence that corresponds toall degenerate variants of the isolated CHIPS gene.

[0029] The invention furthermore relates to nucleic acid molecules thatencode (poly)peptides that do not have the complete sequence of FIG. 5but one or more functional portions thereof that in themselves ortogether constitute a biologically active (poly)peptide having CHIPSactivity. Such portions may vary in size from the complete amino acidsequence minus one amino acid to peptides of at least 2, preferably atleast 5 amino acids. In case the active part of the protein lies in twoor more portions of the complete amino acid sequence, the invention alsorelates to nucleic acid sequences encoding these separate portions in amanner that leads to a peptide configuration that retains the biologicalactivity. In practice this can for example mean that spacer sequencesare to be incorporated in between biologically active portions to leadto a biologically active conformation.

[0030] Thus, when reference is made to “at least part of the sequence”this means not only the three parts described above (i.e. nucleotides1-490, 41-490 and 125-490) but also other fragments of the gene orcombinations thereof provided that they still encode a (poly)peptidehaving CHIPS activity.

[0031] In a further embodiment thereof, the invention thus provides anisolated nucleic acid molecule of the invention which consists of thecoding region of one or more portions of the amino acid sequence of FIG.5, wherein one portion of the amino acid sequence constitutes alone orwith other portions of the amino acid sequence the region(s) of the(poly)peptide having CHIPS activity that lead to biological activity.

[0032] The present invention is not limited to nucleic acid moleculeshaving the exact same sequence as the sequence depicted in FIG. 4 or theabove described variants thereof. Therefore, according to the inventionadditional nucleic acid molecules are provided having a nucleotidesequence which is at least 40%, preferably at least 50%, more preferablyat least 60%, even more preferably at least 70%, most preferably atleast 80%, and the most preferably at least 90% identical to any one ofthe nucleotide sequences as defined under a), b) or c) above.

[0033] It was found that CHIPS is less than 40% homologous to proteinsand peptides known to date. Proteins and peptides that show at least 40%amino acid homology to the CHIPS protein and have CHIPS activity arethus also part of this invention.

[0034] The invention further relates to nucleic acid molecules having anucleotide sequence hybridising under stringent conditions with anucleic acid molecule corresponding with the nucleotide sequence givenin FIG. 4 or degenerate sequences thereof, which encode an amino acidsequence as given in FIG. 5. Stringent conditions are constituted byovernight hybridisation at 42° C. in 5×SSC (SSC=150 mM NaCl, 15 mMtrisodium citrate) and washing at 65° C. at 1.1×SSC. In addition to5×SSC the hybridisation solution may comprise 50% formamide, 50 mMsodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulphateand 20 μg/ml denatured sheared salmon sperm DNA.

[0035] The invention is also not limited to the gene which encodes the(poly)peptide having CHIPS activity, but also relates to nucleic acidmolecules that encode fragments, derivatives and analogues thereof.“Fragments” are intended to encompass all parts of the (poly)peptidethat retain its biological activity. “Fragments” can consist of onesequence of consecutive amino acids or of more than one of suchsequences. “Derivatives” are the complete (poly)peptide having CHIPSactivity or fragments thereof that are modified in some way. Examples ofmodifications will follow hereinbelow. “Analogues” are similar(poly)peptides having CHIPS activity isolated from other organisms, inparticular other pathogenic organisms. All of the above categories haveone thing in common, namely that they have “CHIPS activity”. CHIPSactivity can be measured by any assay that shows directed migration ofleukocytes towards an appropriate chemotactic stimulus. Examples of suchassays include the under agarose technique (as exemplified in Balasoiu,et al., Diabetes care 20: 392-395 (1997)), modified Boyden chambertechniques and transwell systems. The latter technique is furtherillustrated in the examples.

[0036] Therefore, for the present application, the term “(poly)peptideshaving CHIPS activity” is intended to include the original CHIPSprotein, (poly)peptides, fragments, derivatives and analogues thatexhibit CHIPS activity.

[0037] The isolated nucleic acid molecule according to the invention maybe DNA, RNA or cDNA.

[0038] The invention furthermore relates to probes and primers derivedfrom the nucleic acid molecule of the invention. Such primers areoligonucleotides or polynucleotides of at least about 10 consecutivenucleotides (nt), and more preferably at least about 25 nt, still morepreferably at least about 30 nt, and even more preferably about 30-70 ntof the nucleic acid molecule of the invention. Probes are longer and mayfor instance be a portion of the nucleic acid molecule of the inventionof 50-300 consecutive nt, or even as long as the entire nucleic acidmolecule.

[0039] Such oligonucleotides or polynucleotides are useful as diagnosticprobes or as probes in conventional DNA hybridisation techniques or asprimers for amplification of a target sequence by polymerase chainreaction (PCR) as described for instance in Ausubel et al. (supra)

[0040] Furthermore, the invention relates to a recombinant vectorcomprising at least one isolated nucleic acid molecule of the invention.The vector to be used can be selected by the skilled person based on hiscommon general knowledge and will be dependent on the host that is used.

[0041] In addition to vectors, the invention provides for abacteriophage comprising at least one isolated nucleic acid of theinvention. In most CHIPS-positive Staphylococci, the gene encoding CHIPSis located on a prophage and can be turned into an active phage, forexample by treatment with mitomycin according to standard and published,phage isolating procedures. A bacteriophage is thus a useful vehicle tointroduce the CHIPS gene into a host.

[0042] The invention in addition relates to a method for making arecombinant vector, comprising inserting at least one isolated nucleicacid molecule of the invention into a vector. By incorporating more thanone copy in the vector, or introducing more than one vector into a hostthe level of expression can be influenced. When a host cell is used thatcomprises an endogenous gene for a corresponding (poly)peptide havingCHIPS activity, the expression level thereof can be increased byintroducing more copies of the nucleic acid molecule (i.e. the gene)into the host cell or changing the promoter or regulator regions.

[0043] The invention thus also relates to recombinant hosts comprisingat least one isolated nucleic acid molecule or vector of the invention.A number of types of organisms or cells from prokaryotes, protista,fungi, animals or plants may act as suitable host for the expression ofrecombinant (poly)peptides having CHIPS activity. Host cells include thewidely used bacterial strain Escherichia coli including, but not limitedto, the trc expression system (Brosius et al., supra) that allowshigh-level, regulated expression from the trc promotor. Potentiallysuitable other bacterial strains include Gram-positive bacterialstrains, such as Bacillus subtilis, Staphylococcus aureus, or anybacterial strain capable of expressing heterologous proteins. Apreferred production process in E. coli is given in Example 6.

[0044] The (poly)peptide having CHIPS activity may also be produced as arecombinant protein using a suitable expression system employing lowereukaryotes such as yeast or insect cells. Suitable yeast strains includeSaccharomyces cerevisiae, Pichia pastoris, Candida or any yeast straincapable of expressing heterologous proteins. Insect cells used forrecombinant protein expression include the Drosophila system and theBaculovirus system. Alternatively, it may be possible to produce the(poly)peptide having CHIPS activity in an mammalian expression systemthat includes several suitable host cells, including monkey COS cells,hamster CHO, BHK cells or RBL-2H3, human 293, 3T3, HeLa, U937, HL-60 orJurkat cells, mouse L cells and other transformed cells for in vitroculture. For expression of (poly)peptides having CHIPS activity ineukaryotic systems, it may be necessary to modify the protein producedtherein in order to obtain a functional protein. Such modifications,like attachments or substitutions may be accomplished using knownchemical or enzymatic methods. In addition, the sequence of the nucleicacid molecule may be adapted to the codon usage of the host cell.

[0045] The (poly)peptide having CHIPS activity of the invention may alsobe expressed as a product of transgenic animals, e.g. as a component ofthe milk of transgenic cows, goats, pigs, sheep, rabbits or mice whichare characterized by somatic or germ cells containing a nucleotidesequence encoding the (poly)peptide having CHIPS activity.

[0046] The (poly)peptide may be prepared by culturing transformed hostcells under culture conditions suitable to express the recombinantprotein. The resulting protein may then be purified from the culturemedium or cell extracts using a purification process, for examplecomprising the steps of guiding over an absorption chromatography columnthe culture supernatant of the host cell or a liquid obtained therefromafter pre-purification; subsequently guiding the flow-through of theabsorption chromatography column first over an affinity chromatographycolumn and thereafter guiding the eluate of the affinity chromatographycolumn over a DNA column; or subsequently guiding the flow-through ofthe absorption chromatography column first over a DNA column andthereafter guiding the flow-through of the DNA column over an absorptionchromatography column; guiding the flow-through respectively the eluateof the last column of the previous step over a gel filtration column andAnion exchange column, selecting the fraction with a molecular weight ofabout 17 kDa and CHIPS activity. “Flow-through” is herein understood tomean that part of the loaded liquid having situated therein theconstituents which come from the column without extra treatment. Theconstituents in this flow-through do not bind to the column. “Eluate” isunderstood to mean the liquid which comes from the column after elutionand which contains the constituents from the liquid loaded on the columnwhich were bound to the column and were released again therefrom by theelution. In this method the absorption column binds most constituents ofthe loaded culture medium or a liquid obtained therefrom afterpre-purification. The affinity column binds the (poly)peptide havingCHIPS activity and the Snase (Staphylococcal Nuclease) which has asimilar molecular weight as the CHIPS protein and a similar affinity (orlack thereof) for the affinity column respectively the absorptioncolumn. The DNA column binds only the Snase. This method worksparticularly well if the first affinity chromatography column is aso-called Ligand Dye “yellow” column, the second affinity chromatographycolumn is a so-called Ligand Dye “green” column and the DNA column a DNAcellulose column.

[0047] In addition, other known purification methods can be used, suchas gel filtration, ion exchange chromatography, affinity chromatography,hydrophobic interaction chromatography or immunoaffinity chromatography.

[0048] Alternatively the (poly)peptide having CHIPS activity may beexpressed in a form that will facilitate purification. For example, itmay be tagged with a polyhistidine (6×His) epitope and subsequentlypurified by using a resin to which nickel ions are bound by means of achelating agent. The (poly)peptide having CHIPS activity containing thetag is eluted from the resin by lowering pH or by competing withimidazole or histidine. Such epitope is commercially available fromInvitrogen. Introduction of a protease cleavage site, like that forenterokinase, enables removal of the fusion tag to generate maturenative recombinant (poly)peptide having CHIPS activity. Materials andmethods for such an expression system are commercially available fromInvitrogen, using the pTrcHis Xpress™ vectors in combination withProBound™ resin for efficient isolation of His-tagged protein andEnterokinaseMax™ as highly catalytic active protease and EK-Away™enterokinase affinity resin to remove the contaminating presence of theprotease. Other tags known to those skilled in the art that can be usedto facilitate purification include, but are not limited to, glutathion Stransferase (GST fusion), myc and HA.

[0049] The (poly)peptide having CHIPS activity may also be produced byknown chemical synthesis. Methods for constructing polypeptides orproteins by synthetic means are known to those skilled in the art. Thesynthetic protein, by virtue of sharing primary, secondary and tertiarystructural and/or conformational characteristics with the corresponding(poly)peptide having CHIPS activity will posses an activity in commontherewith, meaning CHIPS properties. Thus, such synthetically producedproteins can be employed as biologically active or immunologicalsubstitute for natural purified (poly)peptide having CHIPS activity. Thesynthesis of CHIPS is further illustrated in Example 7.

[0050] The (poly)peptides having CHIPS activity provided herein alsoinclude (poly)peptides characterized by amino acid sequences into whichmodifications are naturally provided or deliberately engineered.Modifications in the (poly)peptide or DNA sequences can be made by thoseskilled in the art using known conventional techniques. Modifications ofinterest in the CHIPS active (poly)peptide sequences may includereplacement, insertion or deletion of selected amino acid residues inthe coding sequence.

[0051] The information contained in the CHIPS protein, its gene andother (poly)peptides having CHIPS activity and their encoding nucleicacid molecules derived therefrom can be used to screen for fragmentsthereof or other agents which are capable of inhibiting or blockingbinding of a (poly)peptide having CHIPS activity to leukocytes, and thusmay act as inhibitors of chemotaxis activity and/or CHIPS binding to itsputative receptor. Appropriate screening assays may for example use thefluorescent labeled purified CHIPS protein that binds to neutrophils asanalyzed by flow cytometry or fluorometry. Example 2 describes such anassay. A suitable binding assay may alternatively employ purified CHIPSreceptor or receptor domain on a carrier with a form of CHIPS protein asligand. Alternatively, an assay can be employed that screens for theability to bind or compete with CHIPS for binding to a specificanti-CHIPS antibody (monoclonal, polyclonal, or single chain antibody)by various immunoassays known in the art, including but not limited tocompetitive and non-competitive ELISA techniques or Biosensor technologyemploying a sensor chip coated with either ligand (CHIPS), antibody orputative CHIPS receptor (Surface Plasma Resonance (SPR) technique likethe BiaCore). Any (poly)peptide having CHIPS activity other than CHIPSmay also be used in the screening assays described. All these methodscan be adapted for High Throughput Screening (HTS).

[0052] Isolated (poly)peptides having CHIPS activity may be usedthemselves as inhibitors of fMLP and C5a binding to their respectivereceptors FPR and C5aR, or to design inhibitors of CHIPS binding, byscreening for competitive inhibition. Inhibitors of CHIPS binding (tothe putative CHIPS receptor or receptor domains) are also useful fortreating such conditions.

[0053] The invention furthermore relates to molecules that are not(poly)peptides themselves but have a structure and function similar tothose of the (poly)peptides described herein. Examples of such moleculesare peptidomimetics. When reference is made in this application to(poly)peptides, it is intended to include also such non-(poly)peptidesthat have a similar or the same structure and function and as aconsequence a similar or the same biological activity as the(poly)peptides.

[0054] The functional activity of CHIPS, the (poly)peptides, theirfragments, derivatives and analogues can be assayed by various methods.Preferentially, this CHIPS activity is measured by its ability toprevent the binding of fluorescent-fMLP (Bodipy-fMLP) or fluorescent-C5a(FITC-C5a) to neutrophils as determined by flow cytometry. Example 1describes such an assay. CHIPS activity is also measured by its abilityto prevent migration of neutrophils towards fMLP or C5a as determined bythe Transwell chemotaxis assay, described in the Examples.Alternatively, an assay based on the ability of chemokines, includingfMLP and C5a, to initiate a rapid and transient rise in intracellularcalcium concentration can be employed to screen for CHIPS activity.Various assays known in the art can be used, including but not limitedto the use of various calcium specific fluorescent probes in combinationwith flow cytometry or fluorometry, or microphysiometry. As cells forthe screening of CHIPS activity by either method, freshly isolatedneutrophils can be used or cells transfected with either FPR or C5aR,wild type or mutated forms of those receptors.

[0055] Isolated (poly)peptides having CHIPS activity may be useful intreating, preventing or ameliorating inflammatory conditions that areinvolved in many diseases and disorders, such as listed in Table 1.Support for the therapeutical usefulness of the (poly)peptides of theinvention for treatment of the diseases in Table 1 can be found in thefollowing references: For ARDS: Demling R H (1995). The modern versionof adult respiratory distress syndrome. Ann. Rev. Med. 46:193-202; andFujishima S, Aikawa N 1995 Neutrophil-mediated tissue injury and itsmodulation. Intensive Care Med 21:277-285; For severe infections(meningitis): Tunkel A R and Scheld W M (1993). Pathogenesis andpathophysiology of bacterial meningitis. Clin. Microbiol. Rev. 6:118.For injury after ischaemia/reperfusion: Helier T, et al. (1999).Selection of a C5a receptor antagonist from phage libraries attenuatingthe inflammatory response in immune complex disease andischemia/reperfusion injury. J. Immunol. 163:985-994. For rheumatoidarthritis: Edwards S W and Hallett M B (1997). Seeing the wood for thetrees: the forgotten role of neutrophils in rheumatoid arthritis.Immunology Today 18: 320-324; and Pillinger M H, Abramson S B (1995).The neutrophil in rheumatoid arthritis. Rheum. Dis. Clin. North Am. 199521:691-714. For myocardial infarction: Byrne J G, Smith W J, Murphy M P,Couper G S, Appleyard R F, Cohn L H (1992). Complete prevention ofmyocardial stunning, contracture, low-reflow, and edema after hearttransplantation by blocking neutrophil adhesion molecules duringreperfusion. J. Thorac. Cardiovasc. Surg. 104:1589-96. For COPD: Cox G(1998). The role of neutrophils in-inflammation. Can. Respir. J. SupplA:37A-40A; and Hiemstra P S, van Wetering S, Stolk J (1998). Neutrophilserine proteinases and defensins in chronic obstructive pulmonarydisease: effects on pulmonary epithelium. Eur. Respir. J. 12:1200-1208.For stroke: Barone F C, Feuerstein G Z (1999). Inflammatory mediatorsand stroke: new opportunities for novel therapeutics. J. Cereb. BloodFlow Metab. 19:819-834; and Jean W C, Spellman S R, Nussbaum E S, Low WC (1998). Reperfusion injury after focal cerebral ischemia: the role ofinflammation and the therapeutic horizon. Neurosurgery 43:1382-1396. Formeningitis: Tuomanen E I (1996). Molecular and cellular mechanisms ofpneumococcal meningitis. Ann. N. Y. Acad. Sci. 797:42-52. TABLE 1Inflammatory conditions as targets for CHIPS SYSTEM DISEASE SYSTEM(cont) DISEASE (cont) cardio- arteriosclerosis genitourinary urinaryvascular tract infection sepsis glomerulonephritis ischaemic shockTrichomonas vaginalis infection cardiopulmonary endometriosis bypassaortic joints rheumatoid arthritis surgery heart acute reactivearthritis transplantation myocardial gout infarction central bacterialrespiratory ARDS meningitis nervous viral COPD meningitis multipleidiopathic pulmonary sclerosis fibrosis stroke cystic fibrosisAlzheimer's asthma disease Brain pleural emphema tumour gastro-pencreatitis metal fume fever intestinal ulcerative bacterial pneumoniacolitis Crohn's chronic bronchitis disease alcoholic hypersensitivitypneumonia hepatitis viral Mycobacterium tuber. hepatitis infectionHeliobacter viral respiratory tract pylori infection gastritis gastricallergic rhinitis carcinoma hepatocellular sinusitis carcinomaperitonitis bronchogenic carcinoma skin psoriasis various periodontitiscontact HIV infection dermatitis atopic chronic lymph dermatitisleukemia cutaneous acute transplant rejection T-cell lymphoma burnsglomerulonephritis frost bite repetitive strain injury

[0056] According to a further aspect thereof, the invention thus relatesto (poly)peptides having CHIPS activity for use in diagnosis,prophylaxis or therapy, in particular for use in the treatment of acuteand chronic inflammation reactions and HIV infection, more in particularfor use in the treatment of Adult Respiratory Distress Syndrome (ARDS),ischaemic shock, traumatic brain injury, severe infections, myocardialinfarction, stroke, vessel surgery, ulcerative colitis, Crohn's disease,Chronic Obstructive Pulmonary Disease (COPD), rheumatoid arthritis,dermatoses, multiple sclerosis, Alzheimer's disease, arteriosclerosis,repetitive strain injury (RSI), acute transplant rejection, burns, acutereactive arthritis, pancreatitis, vasculitis, glomerulonephritis, gout,frost bite and meningitis.

[0057] The invention furthermore relates to the use of the(poly)peptides having CHIPS activity for the manufacture of apreparation for diagnosis, prophylaxis or therapy, in particular for thetreatment of acute and chronic inflammation reactions and HIV infection,more in particular for the treatment of the indications listed above.

[0058] Also part of the present invention are therapeutic compositionscomprising a suitable excipient and the (poly)peptide having CHIPSactivity of the invention. Such composition can be used for thetreatments as specified above.

[0059] The invention further relates to use of the nucleic acid moleculeof the invention, optionally incorporated in a larger construct, forvarious purposes, such as raising antibodies thereto, modulating theCHIPS activity or in a therapeutic preparation.

[0060] The invention further relates to nucleic acid molecules and theamino acid sequence encoded by the nucleic acid molecules that can beidentified by so-called “computer cloning”. More specifically, thistechnique comprises using (1) the nucleic acid sequence as depicted inFIG. 4, or fragments, derivatives and analogues thereof, or (2) theamino acid sequence as depicted in FIG. 5, or fragments, derivatives andanalogues thereof, as a query for screening nucleic acid sequences ornucleic acid sequence databases, or protein sequences or proteinsequence databases, using search algorithms that can identify regionswith homology. Such algorithms are known to the person skilled in theart and include, but are not limited to, BLAST searches (Altschul etal., J. Mol. Biol. 215, 403-410 (1990)). The sequence databases that maybe searched include, but are not limited to, the Genbank™ database andthe Swissprot™ database. When using a BLAST search or modificationsthereof, generally subjects that display homology can be identified,Identification is based on the value of the Score or the Smallest SumProbability P(N). Homologues of the CHIPS nucleic acid sequence or(poly)peptide sequence are defined by a Score that is at least 200,preferably at least 400, more preferably at least 800, most preferablyat least 1600. Alternatively, the P(N) value can be used foridentification of homologous sequences. Homologues of the CHIPS nucleicacid sequence or (poly)peptide sequence are defined by a P(N) value thatis smaller than 1e-3, preferably smaller than 1e-6, more preferablysmaller than 1e-12, even more preferably smaller than 1e-24, mostpreferably smaller than 1e-48.

[0061] In a still further embodiment of the invention antibodies orbiologically active fragments thereof specifically directed to the(poly)peptide of the invention and CHIPS-based, CHIPS receptor-blockingmolecules are provided. Such CHIPS-based, CHIPS receptor-blockingmolecules, and antibodies or biologically active fragments thereof andchimerics, single chains, and expression libraries may be used toneutralise the activity of the CHIPS protein or related (poly)peptidesin prophylaxis or therapy, or may be used for diagnostic purposes tobind CHIPS or related (poly)peptides. Such antibodies and CHIPS-based,CHIPS receptor-blocking molecules are for example useful for thetreatment of Staphylococcus infection. The invention also providestherapeutic compositions comprising a suitable excipient and one or moreof these antibodies and/or biologically active fragments thereof.

[0062] “CHIPS-based, CHIPS receptor-blocking molecules” are moleculesthat compete with CHIPS in a CHIPS binding assay as described in Example8. Such “CHIPS-based, CHIPS receptor-blocking molecules” may or examplebe molecules that have the same amino acid composition and amino acidsequence as CHIPS, but not the complete sequence. Such molecules can besingle fragments of CHIPS, or may consists of multiple CHIPS fragments,all still having CHIPS activity. However, all other molecules that meetthe functional requirement of competing with CHIPS in a CHIPS bindingassay are also included.

[0063] The isolated nucleic acid molecules of the invention canfurthermore be used for gene therapy. The nucleic acid molecule can beintroduced at the site of inflammation to act locally or at a distantsite. Gene therapy is via viral vectors, such as, but not limited to,adenoviral vectors, adeno-associated viral vectors or lentiviralvectors. Alternatively, non-viral vectors, such as those based onliposomes or polymers may be used. Gene therapeutic strategies are basedon (1) in vivo gene therapy, where the isolated nucleic acid moleculesof the invention are introduced into target cells in vivo, or (2) exvivo gene therapy, where the isolated nucleic acid molecules of theinvention are introduced into target cells ex vivo, followed byadministration of the transduced cells, or a subpopulation of thetransduced cells, into an individual.

[0064] The invention relates to a method for treating a subjectsuffering from inflammation by administering a therapeutically effectiveamount of a (poly)peptide of the invention and a method for genetherapeutically treating a subject suffering from inflammation byadministering a therapeutically effective amount of a nucleic acidmolecule, as well as a method for treating a subject suffering fromstaphylococcus infection by administering a therapeutically effectiveamount of an antibody and/or biologically active fragment thereof.

[0065] The nucleic acid molecules of the invention can be used in amethod for isolating from an organism a gene encoding a protein havingCHIPS activity, which method comprises screening of a genomic or cDNAlibrary of that organism with a probe based on the nucleic acidmolecule, and isolation of the positive clones.

[0066] According to a further aspect thereof, the invention relates tomicro-organism harboring one or more nucleic acid molecules of theinvention for use as a medicament for the treatment of acute and chronicinflammation reactions and HIV infection, in particular for treatingAdult Respiratory Distress Syndrome (ARDS), ischaemic shock, traumaticbrain injury, severe infections, myocardial infarction, stroke, vesselsurgery, ulcerative colitis, Crohn's disease, Chronic ObstructivePulmonary Disease (COPD), rheumatoid arthritis, dermatoses, multiplesclerosis, Alzheimer's disease, arteriosclerosis, repetitive straininjury (RSI), acute transplant rejection, burns, acute reactivearthritis, pancreatitis, vasculitis, glomerulonephritis, gout, frostbite and meningitis.

[0067] The invention further relates to a diagnostic PCR test forscreening a patient infected with Staphylococcus aureus on the presenceof the CHIPS gene. CHIPS is an important staphylococcal virulencefactor, so patients with CHIPS containing Staphylococci are at higherrisk for invasive diseases and might need different or additionaltreatment.

[0068] All molecules of the invention, i.e. nucleic acid molecules,(poly)peptides, non-(poly)peptides, fragments, derivatives andanalogues, may find various other applications. Such applicationsinclude, but are not limited to:

[0069] Isolation of factors that can bind the above mentioned molecules.Examples of such factors being receptors and proteins. Such isolationcan for instance be performed using the yeast two hybrid system or usingtagged molecules of the invention as bait for fishing.

[0070] Design of peptoids and peptidomimetics.

[0071] Making phage display libraries, which can in turn be used fordetermining active domains, functional equivalents etc.

[0072] Identifying signal transduction pathways that are activated orinactivated by CHIPS and the molecules of the invention.

[0073] Assay for determination of the biological CHIPS activity(chemotaxis inhibition or chemokine receptor expression)

[0074] All molecules of the invention can be labeled in any way.Examples of labeling include but are not limited to fluorescence,biotin, radioactive labeling etc. Such labeled molecules can be used forscreening of compounds that resemble or overlap with the biologicalactivity of CHIPS, as well as identification of binding sites, both invivo and in vitro, and for tracing CHIPS protein or nucleic acid in anorganism.

[0075] It is clear that where reference is made herein to a(poly)peptide having a particular amino acid sequence, it is intended toalso encompass (poly)peptides containing one or more amino acids thatare chemically modified in a manner obvious to one skilled in the art,provided that such modification does not abolish the CHIPS activity.

[0076] The present invention will be further illustrated in the examplesthat follow and that are in no way intended to be limiting to thisinvention. In this description and the examples reference is made to thefollowing figures and tables:

[0077]FIG. 1 shows the CHIPS activity of the eluate from the Mono Qcolumn.

[0078]FIG. 2 shows the Coomassie Blue stained SDS-PAGE of purified CHIPSafter the final Mono Q chromatography step.

[0079]FIG. 3 shows the concentration dependent binding of CHIPS-FITC tothe various leukocyte populations.

[0080]FIG. 4 shows the sequence of the chp gene from S. aureus Newman.The Shine Dalgarno sequence (AGGAGA) and the chp open reading frame(ORF) are under-lined. The nucleotides encoding the mature protein areindicated by a double line. Diverging nucleotides in S. aureus 1690sequence are indicated above the sequence.

[0081]FIG. 5 shows the amino acid sequence deduced from the S. aureusNewman chp gene. The region matching the N-terminal 35 amino acids ofCHIPS is underlined. Diverging amino acids in the S. aureus 1690 proteinare indicated above the sequence.

[0082]FIG. 6 shows the detection of the chp gene in the genomes of S.aureus strains.

[0083]FIG. 7 shows CHIPS activity in the supernatants of S. aureusstrains.

[0084]FIG. 8 shows the distribution of the chp gene in the genomes ofvarious clinical S. aureus strains.

[0085]FIG. 9 shows two dose response curves of rabbit anti-CHIPSantibodies binding to CHIPS derived peptides of amino acids 1 through 15(FIG. 9A) and purified CHIPS (FIG. 9B) as determined by ELISA.

[0086]FIG. 10 shows the concentration dependent inhibition of neutrophilmigration towards fMLP by purified CHIPS, expressed as percentage ofbuffer treated cells. Cells were incubated with various concentrationsCHIPS for 30 min at room temperature and added to the upper compartmentof the Transwell container. Migration towards 1×10⁻⁸ M fMLP wasdetermined after 60 minutes incubation at 37° C.

[0087]FIG. 11 is a representative image of an SDS-PAGE showing the finalpurified recombinant CHIPS (rCHIPS) obtained from an E. coli lysateafter affinity chromatography over a Nickel column and cleavage of theHistidin tag by Enterokinase.

[0088]FIG. 12 shows the concentration dependent inhibition ofrecombinant CHIPS (rCHIPS) on the expression of the receptor for fMLP(FPR) and C5a (C5aR) on neutrophils.

[0089]FIG. 13 shows the concentration dependent impairment of theintracellular free calcium release induced by fMLP and C5a inneutrophils.

[0090]FIG. 14 shows the concentration dependent inhibition of CHIPS-FITCbinding by the complete recombinant CHIPS and the recombinant mutantCHIPS⁴⁻¹²¹.

[0091] Table 1 shows inflammatory conditions that can be treated withthe (poly)peptides and non-(poly)peptides of the invention; and

[0092] Table 2 shows the binding in ELISA of several selected clones ofmonoclonal antibodies derived from a mouse immunized with CHIPS. Bindingis to purified CHIPS and the reacting mouse monoclonals are detectedwith a HRPO-coupled anti-mouse antibody.

EXAMPLES Example 1 Purification of CHIPS Protein From S. aureusSupernatant Material and Method

[0093] 1.1 Isolation of the Protein Staphylococcus aureus 1690 (aclinical isolate, University Medical Center Utrecht (UMC Utrecht)) orStaphylococcus aureus Newman (a gift from Dr Foster, Dublin) is culturedovernight in IMDM medium (Gibco) and subsequently diluted 1:40 in freshIMDM for a 7 hour culture at 37° C. After pelleting of the bacteria theS. aureus supernatant (referred to as SaS) is collected, filtered over a0.2 μm filter and immediately used further (Veldkamp et al.,Inflammation 21. 541-551 (1997). A quantity of 5 liters of SaS is guidedover three columns (25 ml) coupled in tandem. These three columns aresuccessively a “Reactive Yellow” 861, ligand dye cross-linked 4% beadedagarose column (Sigma), a DNA Cellulose (Pharmacia) and a “ReactiveGreen” 19 ligand dye cross-linked 4% beaded agarose column (Sigma).

[0094] After washing with PBS the green (Reactive Green 19 column iseluted with 2 M NaCl and the second 50 ml, containing CHIPS activity, ispooled. PMSF (1 mM) is added and the eluate is dialysed in PBS for 18hours. The sample is concentrated to a volume of ±10 ml in a dialysisbag soaked in polyethylene glycol. The concentrated material isseparated on a Pharmacia Superdex-200 gel filtration column, whereafterthe active fractions (4 ml volumes) are pooled, treated with PMSF (1 mM)and dialysed in 10 mM Tris-HCl (pH 8.0) for 18 hours. The pooled activefractions are loaded onto a Mono Q anion exchange column (Pharmacia)that is eluted with a gradient of 10 mM Tris-HCl buffer ranging from 0to 1 M NaCl. Active fractions (1 ml volumes) are pooled and used as thefinal preparation of purified CHIPS. Protein content is determined witha Pierce Micro-BCA assay and CHIPS is stored at −20° C. in smallaliquots. The final isolated material is analysed for purity on a 12.5%SDS-PAGE (Mini-Protean II; BioRad) after staining with Coomassie Blue.The CHIPS protein appears as a single band with an apparent molecularweight around 17 kDa. All fractions are screened for CHIPS activity byits capacity to inhibit binding of fluorescent-labeled fMLP to isolatedneutrophils as measured by flow cytometry.

[0095] 1.2 Binding of fMLP and C5a to Granulocytes

[0096] Granulocytes are isolated from heparinized blood of healthyvolunteers via a Histopaque-Ficoll gradient in accordance with thestandard method (Troelstra et al., J. Leukocyte Biol. 61, 173-178(1997)). The remaining erythrocytes in the granulocyte fraction arelysed with sterile water (for 30 sec.) and washed after recovery of theisotonicity. The cells are finally resuspended in PRMI (Gibco) with0.05% Human Serum Albumin (RPMI/HSA). In Falcon tubes 50 μl cells (5×10⁶cells/ml) are incubated with 50 μl CHIPS-containing material (SaS,purified CHIPS or column fractions) for 30 min at 37° C. The cells areplaced on ice and washed once with RPMI/HSA (at 4° C.) and resuspendedin 50 μl fresh medium. 5 μl BODIPY-labeled fMLP (final concentration 0.1μM; Molecular Probes) or FITC-labeled C5a (final concentration 1 μM;recombinant C5a from Sigma, labeled with FITC as described in example2.1 for CHIPS) is then added and the sample is incubated for 60 minuteson ice. After washing the fluorescent fMLP or C5a binding to thegranulocytes is analysed with a flow cytometer (FACScan; BectonDickinson). The average fluorescence value of 5000 granulocytes iscalculated with Lysis II software.

Results

[0097]FIG. 1 shows the elution profile (OD280) of the CHIPS activity ofthe eluate from the Mono Q column. The volume fractions between 39 and41 ml show the strongest CHIPS activity. FIG. 2 shows the Coomassie Bluestained SDS-PAGE of purified CHIPS after the final Mono Q chromatographystep.

Example 2 Specific Binding of CHIPS to Neutrophils and MonocytesMaterial and Method

[0098] 2.1 FITC Labeling of Purified CHIPS Protein

[0099] Purified CHIPS (500 μg/ml protein) is incubated with {fraction(1/10)}th volume of 1 mg/ml FITC (Fluorescein Isothiocyanate, Isomer I;Sigma) in a 1 M Sodium carbonate buffer pH 9.6 for 1 hour at roomtemperature. FITC-labeled CHIPS is separated from free FITC by passingthe mixture over a desalting column (Pharmacia, Fast Desalting HR 10/10)and monitoring the eluate for OD₂₈₀ and fluorescence by an on-linecoupled fluorometer (Perkin Elmer). Fractions with high OD₂₈₀ andfluorescence were pooled and analyzed for protein content with the MicroBCA protein assay (Pierce). CHIPS-FITC is stored in small aliquots at−20° C.

[0100] 2.2 Binding of CHIPS-FITC to Leukocytes.

[0101] The specific binding of CHIPS-FITC to leukocytes is determined byflow cytometry. Purified neutrophils and mononuclear cells (consistingof monocytes and lymphocytes) are isolated from heparinized blood ofhealthy volunteers as described (Troelstra et al., Infect. Immun. 65:2272-2277 (1997)). Isolated cells are remixed to obtain a ratio of cellsthat mimics the situation in blood. Human red blood cells are obtainedby washing a small aliquot of whole blood thrice with PBS. Theconcentration of red blood cells is determined photospectrometrically.

[0102] In Falcon tubes 50 μl leukocytes or red bloodcells (5×10⁶cells/ml) are incubated with 5 μl CHIPS-FITC at various concentrationsfor 30 min on ice. Cells are washed once with medium (RPMI containing0.05% HSA) and resuspended in 150 μl fresh medium. Binding of CHIPS-FITCto the leukocyte is measured by flow cytometry (FACScan; BectonDickinson). Association with the various subpopulations is analyzed byselective electronic gating on forward (FSC) and sideward (SSC) scatterparameters in LysisII software (BD). The average fluorescence value ofthe selected cell population is calculated with the software.

Results

[0103]FIG. 3 shows the concentration dependent binding of CHIPS-FITC tothe various leukocyte populations. It can be seen that CHIPS-FITC bindsmost efficiently to neutrophils, followed by monocytes. CHIPS-FITC doesnot bind to red blood cells and marginally to lymphocytes, but only to asubpopulation. Binding of CHIPS-FITC to neutrophils is specific becauseaddition of a 10-fold excess of non-fluorescent labeled CHIPS completelyinhibits association of CHIPS-FITC to the cells.

Example 3 Secuence, Cloning, and Expression of the CHIPS-Encoding Gene(chp) of Staphylococcus aureus Material and Method

[0104] 3.1 Bacterial Strains, Plasmids and Growth Conditions

[0105]Staphylococcus aureus Newman, RN4220, and COL are commonly usedlaboratory strains. S. aureus 1690 is a clinical strain, isolated from apatient with bacteremia (K. E. Veldkamp et al., Inflammation, 21:541-551(1997)). Escherichia coli DH5α was used as a cloning host (F. M. Ausubelet al., Current Protocols in Molecular Biology, John Wiley and Sons,Inc., New York, N.Y. (1990)). Plasmid pRB474 is a shuttle vector for E.coli and staphylococci containing the veqII promoter from Bacillussubtilis that permits expression of genes cloned into the multiplecloning site of pRB474. pRB474 is a derivative of pRB374 (R. Brückner,Gene, 122:187-192 (1992)) in which the neomycin resistance gene has beenreplaced by a chloramphenicol resistance gene. All strains were grown inBM broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% K₂HPO₄, 0.1%glucose) at 37° C. unless otherwise noted.

[0106] 3.2 Sequence Analysis

[0107] DNA was sequenced by cycle sequencing on a DNA sequencer 4000 L(LI-COR Inc., Lincoln, Nebr., USA) using the Thermo Sequenase™fluorescent-labeled prime cycle sequencing kit (Amersham, LittleChalfont, UK). Suitable primers were used to directly sequence genomicDNA which was isolated according to J. Mamur (J. Mol. Biol., 3:208-218(1961)). The sequencing method has been described briefly in Peschel etal. (J. Biol. Chem., 274:8405-8410 (1999)). To perform sequencesimilarity searches, the program BLAST 2.0 with the non-redundantprotein database of the NCBI,(Bethesda, Md., USA) was used. Sequencealignments were accomplished using the Higgins-Sharp algorithm of theprogram MacDNASIS Pro (Hitachi Software Engineering, San Bruno, Calif.,USA).

[0108] Previously, the first 35 amino acids of CHIPS have beendetermined by N-terminal sequencing of the purified protein. The S.aureus DNA is very rich in A and T nucleotides while G and C nucleotidesare rare (only about 30% of total bases). Thus, for most amino acids,the most A- and T-rich codons are preferred. According to this rule, aprimer sequence was derived from amino acids 15-24(GAAAAAGAAAAAGCATATAAAGAA (SEQ ID NO 1). The primer was used to directlysequence genomic DNA from S. aureus Newman yielding a sequence ofseveral hundred base pairs. A new primer was derived from this sequenceto read toward the binding site of the first primer. The combined DNAsequence contained the binding site of the first primer with twodifferences (G instead of A in position 3 and T instead of A in position15) (FIG. 4). It encoded an open reading frame of 450 bp preceded by areasonable Shine Dalgarno sequence for initiation of translation (J.Shine and L. Dalgarno, Proc. Natl. Acad. Sci. USA, 71:1342-1346 (1974))and followed by three stop codons. The gene was named chp; it encodes aputative protein of 149 amino acids with no similarities to any proteinin the databases. The N-terminal 28 amino acids seem to form a signalpeptide for secretion across the cytoplasmic membrane (3 positivelycharged residues followed by a non-charged region of 22 amino acids andan ALA-X-ALA consensus motive for cleavage by the signal peptidase 1;FIG. 5) (G. von Heijne, Nucl. Acids Res. 14:4683-4690 (1986)). Thesignal peptide is followed by a region that matches almost perfectly theN-terminal 35 amino acids of CHIPS. The only exception is a serine inposition 33 of the deduced mature protein instead of an asparagineresidue predicted by N-terminal sequencing. The deduced mature proteinhas a size of 121 amino acids and 14.1 kDa and an isoelectric point of9.32. It thus fulfills all requirements for the CHIPS protein. Using thesame primers, the chp gene of S. aureus 1690 was sequenced. The twogenes were almost identical with 5 deviations. On amino acid sequencelevel, only one position was exchanged (FIGS. 4 and 5).

[0109] 3.3 Cloning and Expression of the chp Gene

[0110] The chp gene from S. aureus Newman was amplified by PCR usingprimers whose sequence was modified to introduce restriction sitespermitting the cloning of chp in the expression plasmid pRB474. Theresulting plasmid pPr4-chp contained the chp coding region, 19 bpupstream from the start codon containing the Shine Dalgarno sequence and104 bp downstream from the first stop codon. The fragment was insertedin the appropriate orientation permitting expression of the gene by theveqII promoter and the identity of the fragment was verified by sequenceanalysis. Plasmid pPr4-chp was transferred to the restriction-negativestrain S. aureus RN4220 by electroporation (J. Augustin and F. Götz,FEMS Microbiol. Lett. 66:203-208 (1990)), isolated from a positiveclone, and electroporated into S. aureus COL (TIGR accession no. 1280).The identity of the plasmid was verified by restriction fragmentanalysis and sequencing of the insert.

[0111] The chp gene was not contained in the partly available genomesequence of S. aureus COL (TIGR accession no. 1280). By PCR analysis itwas demonstrated, that the gene is in fact lacking in S. aureus COLwhile S. aureus Newman and 1690 were positive (FIG. 6). Furthermore, S.aureus COL was negative in the CHIPS activity assay (FIG. 7). The chpgene from S. aureus Newman was cloned in plasmid pPr4-chp, which permitsexpression of cloned genes by a plasmid-encoded promoter. Transformationof S. aureus COL with the plasmid rendered the strain positive in theCHIPS assay (FIG. 7), proving that the chp gene encodes the CHIPSprotein.

3.4 Detection of the chp Gene by PCR

[0112] The absence or presence of the chp gene in various S. aureusstrains was determined by PCR using crude cell extracts as a templatesource. One bacterial colony from a fresh agar plate was resuspended in1.5 ml saline, sedimented, and resuspended in100 μl of a lysis mixsolution containing 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mglysostaphin/ml, and 0.1 mg achromopeptidase/ml. Samples were incubatedat 37° C. for 30 min and then centrifuged. The clear supernatant washeated to 100° C. for 5 min and subsequently diluted by addition of 400μl TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8). 1 μl of the cellextracts were applied to PCR reactions using the chp-specific primerschp-5′ (GAAAAAGAAATTAGCAACAACAG (SEQ ID NO 2)) and chp-3′(CATAAGATGATTTAGACTCTCC (SEQ ID NO 3). Amplification was accomplished by35 cycles composed of 1 min at 90° C., 1 min at 55° C., and 1 min at 72°C. The resulting PCR product comprised 90.4% of the chp gene starting 2bp downstream of the start codon and ending 41 bp upstream of the firststop codon. The PCR products were subjected to agarose gelelectrophoresis. All sequencing, PCR, and recombinant DNA techniqueswere carried-out according to standard procedures (F. M. Ausubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NewYork, N.Y. (1990)).

[0113] 3.5 Assay for CHIPS Activity

[0114]S. aureus strains were analyzed for CHIPS activity in an assay forbinding of fluorescence-labeled fMLP to human neutrophils. Strains werecultivated in IMDM medium (Life Technologies, Paisley, UK), for 24 h andculture supernatants were dialyzed and tested as described in example1.2.

Results

[0115]FIG. 4 shows the sequence of the chp gene from S. aureus Newman.The Shine Dalgarno sequence (AGGAGA) and the chp open reading frame(ORF) are underlined. The nucleotides encoding the mature protein areindicated by a double line. Diverging nucleotides in S. aureus 1690sequence are indicated above the sequence.

[0116]FIG. 5 shows the amino acid sequence deduced from the S. aureusNewman chp gene. The region matching the N-terminal 35 amino acids ofCHIPS is underlined. Diverging amino acids in the S. aureus 1690 proteinare indicated above the sequence.

[0117]FIG. 6 shows the detection of the chp gene in the genomes of S.aureus strains. PCR products obtained with chp-specific primers wereseparated on an agarose gel. Lanes 1 and 2, S. aureus Newman; lanes 3and 4, S. aureus COL; lanes 5 and 6, S. aureus 1690. The followingbacteria were found to be negative for the presence of the chp gene asdetermined by PCR: Staphylococcus capitis, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus epidermidis, Staphylococcussaprophyticus, Staphylococcus warneri and Escherichia coli.

[0118]FIG. 7 shows CHIPS activity in the supernatants of S. aureusstrains. Various concentrations of culture supernatants of S. aureus1690 (squares), COL wild-type (open circles) and COL with plasmidpPr4-chp (solid circles) were tested for inhibition of fMLP binding tohuman neutrophils. The background fluorescence was subtracted and valuesare given as % of the control samples (incubation without culturesupernatants).

[0119]FIG. 8 shows the distribution of the chp gene in the genomes ofvarious clinical S. aureus strains. Bacteria are screened by PCR withchp-specific primers and evaluated for the presence of the specific 400bp band on an agarose gel. S. aureus strains are grouped on focus ofisolation from the patients. Lab Laboratory strains; Others=strains fromother body fluids; CAPD=Chronic Ambulatory Peritoneal Dialysis cultures;Blood=blood cultures; Wound=wound infections; MRSA=Multiple ResistanceS. aureus strains.

Example 4 Antibodies Specific for CHIPS Material and Method

[0120] 4.1 Immunization

[0121] Antibodies specific for CHIPS protein can be produced usingpurified natural or recombinant protein or sequence derived syntheticpeptides, as antigen. Both polyclonal and monoclonal antibodies havebeen produced using standard techniques (as described in Harlow and Lane(1988), Antibodies, A Laboratory manual, Cold Spring Harbor LaboratoryPress; and Erich, et al (1989), J. Immunol. 143: 4053-4060). On thebasis of the first 15 amino acids, a synthetic peptide was made inaccordance with standard Fmoc chemistry as described in De Haas et al.,J. Immunol. 161:3607-3615. The peptide was coupled to Keyhole LimpetHemocyanin in accordance with the instructions of the manufacturer(Pierce) and subcutaneously immunized with Freund's Complete Adjuvant,followed by two booster injections with Freund's Incomplete adjuvant.

[0122] Immunoglobulins from the sera of immunized animals or hybridomacell culture supernatants are isolated by affinity chromatography usingcommercial resins containing Protein A, Protein G or recombinationsthereof (Pharmacia).

[0123] 4.2 ELISA

[0124] Antisera and purified antibodies (IgG) are screened forreactivity with purified CHIPS protein or derived synthetic peptides byELISA. Therefor the antigen is coated on a microtitre plate (Nunc‘Maxisorb’) in a concentration of 1 to 3 μg/ml in a 0.1 M carbonatebuffer pH 9.6 during 18 hours at 4° C. After washing, non occupiedplastic is blocked with 4% BSA in PBS/Tween 20 (0.05) for 1 hr at 37° C.Serial dilutions of the antibodies are made in PBS/Tween containing 2%BSA and incubated for 1 hr at 37° C. Bound antibodies are incubated witha 1/5000 diluted peroxidase labeled secondary antibody, either goatanti-rabbit IgG for polyclonal antibodies or goat anti-mouse IgG formonoclonal antibodies (both from Southern Biotechnology Associates,Inc.), for 1 hr at 37° C. Reactions are developed with TMB as substrateand the Optical Density (OD) was read at 450 nm.

Results

[0125]FIG. 9 shows the specific binding of polyclonal IgG from a rabbitimmunized with a synthetic peptide comprising the first 15 N-terminalamino acids of CHIPS (anti-CHIPS-peptide). A high OD₄₅₀ is shown for therabbit anti-CHIPS-peptide with the peptide (FIG. 9A) as well as thepurified CHIPS protein (FIG. 9B) coated to the wells. The effect isconcentration dependent and is already significant with a minimalconcentration of 30 ng/ml IgG. A non-immunized pool of normal rabbit IgGgives some background binding, but only at high antibody concentrations,especially when purified CHIPS is coated to the ELISA plate.

[0126] Table 2 shows the specific binding of selected hybridoma clonesderived from mice immunized with purified CHIPS protein. TABLE 2 clonename OD₄₅₀ background 0.056 25-1 0.973 25-2 0.985 25-3 1.286 29-1 1.84729-2 1.433 29-3 1.564 29-4 2.123

Example 5 Chemotaxis Assay

[0127] The CHIPS activity of (poly)peptides and non-(poly)peptides ofthe invention can for example be determined with the following assay.

[0128] In order to determine the directed migration use is for examplemade of a Transwell system (Costar) consisting of an upper compartmentand a lower compartment separated by a 3 μm polycarbonate membrane. Thegranulocytes are labelled with ECECF (2-carboxyethyl-5-(and-6-)carboxyfluorescein; Molecular Probes), a fluorescent label which entersthe cytoplasm of the cells. The cells (5×10⁶) are incubated for 20minutes at 22° C. with 3 μM BCECF-AM (the acetomethyl ester of2-carboxyethyl-5-(and-6-)-carboxyfluorescein), subsequently washed threetimes and resuspended to 5×10⁶ cells/ml in RPMI/HSA. 100 μl of cells andthe desired quantity of the CHIPS protein is introduced into the uppercompartment of the Transwell system and the whole is suspended in thewells of a standard 24-well microtitre plate (Costar). Each wellcontains 600 μl RPMI/HSA with or without addition of the chemoattractantfor testing. The chemoattractants are: recombinant C5a (Sigma),recombinant interleukin-8 (Pepro Tech), Platelet Activating Factor-16(PAF-16; Calbiochem) or fMLP (Sigma). After 60 minutes incubation at 37°C. the Transwell container is lifted from the wells and the microtitreplate is analysed for fluorescence in a CyoFluorII(PerSeptiveBiosystems). The degree of fluorescence is a direct measurefor the number of granulocytes which has migrated through the membraneand is expressed as a percentage of the fluorescence of the added numberof cells.

Results

[0129]FIG. 10 shows the concentration dependent inhibition of neutrophilmigration towards FLMP by purified CHIPS, expressed as percentage ofbuffer treated control cells.

Example 6 Production of Recombinant Polypeptide Having CHIPS Activity inE. coli

[0130] CHIPS was produced in E. coli and was found to be biologically asactive as the naturally occurring CHIPS from S. aureus.

[0131] The production method used for the production of recombinantCHIPS can also be used for other (poly)peptides having CHIPS activity.This production method is illustrated hereinbelow.

[0132] The DNA sequence for CHIPS from S. aureus is cloned into asuitable vector that enables efficient expression of CHIPS in competentE. coli host cells using conventional molecular biology techniques. Thestrategy used enables expression of the complete CHIPS protein linked toa removable HIS-tag at the N-terminus in the cytoplasm of E. coli. Thetrc Expression System (pTrcHIS B vector; Invitrogen) was used thatenables expression of non-toxic proteins in E. coli. This system usesthe trc promotor for high-level, regulated expression in any E. colistrain with a multicloning vector. The vector contains an N-terminalpolyhistidine (G×His) tag for rapid purification, a Xpress epitope foreasy detection with an anti-Xpress antibody and an Enterokinase cleavagesite for removal of fusion tag.

[0133]S. aureus Newman chromosomal DNA was used as template for the PCRreaction using Pwo-DNA polymerase that results in a blunt ended PCRproduct. The primers used are CHIPS-TTT (starts exactly with the firstamino acid of CHIPS (F) and CHIPS-TAA (containing a stop codon and aEcoRI-site).

[0134] The PCR product is digested with EcoRI and the pTrcHIS B vectorwith BamHI. The 5′ overhang is removed with S1-nuclease to make theBamHI site blunt ended exactly where the enterokinase (EK) will digestthe protein. Thereafter the vector is digested with EcoRI and ligatedwith the digested PCR product.

[0135] For transformation of the vector, TOP-10 E. coli is used(InVitroGen) using standard calcium precipitation (F. M. Ausubel et al.,1990, Current Protocols in Molecular Biology, John Wiley and Sons, Inc.,New York, N.Y.). Clones are screened on ampicillin containing plates andproper ligation of CHIPS gene is verified by equencing of the isolatedplasmid (clone-29).

[0136] After expression of the CHIPS gene, the E. coli bacteria arelysed and the protein mixture is applied onto a Nickel-ion affinitycolumn (ProBond). Therefore a culture of clone29 in LB medium +50 μg/mlampicillin is initiated with 1 mM IPTG for 4 h at 37° C. Bacteria arecentrifuged and the pellet resuspended in cold phosphate buffer pH 7.8and stored at in −20° C. For cell lysis, lysozyme (100 μg/ml) is addedfor 15′ on ice, tubes are sonicated, frozen in liquid N₂ and thawed in a37° C. waterbath. This cycle of sonication/freeze/thaw is repeated 3times.

[0137] Thereafter RNase and DNase (5 μg/ml) are added for 30′ on ice.The mixture is centrifuged at 3000 g for 30′ at 4° C. and filteredthrough a 0.45 μm filter. The final lysate is diluted 1:1 with coldPhosphate buffer pH 7.8 and run through a charged Nickel column(InVItroGen). The column is washed with phosphate buffer pH 7.8, withphosphate buffer pH 6.0 and with phosphate buffer pH 5.3. The boundCHIPS is eluted with 500 mM imidazole in pH 6.0 phosphate buffer.

[0138] The HIS-tag is removed by enterokinase cleavage followed byremoval of the protease with an EK-Away enterokinase affinity resin.Therefore the eluate is dialysed overnight in cold digestion buffer (50mM Tris-HCl, 1 mM CaCl₂ and 0.1% Tween-20, pH 8.0), filtered through a0.45 μm filter and digested with 0.175 μl Enterokinase/ml HIS-CHIPSproduct. This amount of Enterokinase is batch-dependent and results in apartial digestion to avoid the generation of breakdown products. Thedigested product is. dialysed against phosphate buffer pH 7.8 and passedover a fresh Nickel column to eliminate uncleaved His-tagged CHIPS(HIS-CHIPS); the run through is pure recombinant CHIPS (rCHIPS).Undigested HIS-CHIPS can be eluted again from Nickel column for a seconddigestion round. The Nickel column is finally washed with 50 mM EDTA,0.5 M NaOH, water, 5 mg/ml NiCl₂, water and stored in 20% ethanol.

[0139] All steps in the isolation and digestion of HIS-CHIPS are.checked by SDS-PAGE on a 16.5% Tris-Tricine Ready gel (BioRad). Samplesare mixed 1:1 with sample buffer (200 mM Tris-HCl pH 6.8, 2% SDS, 40%glycerol, 0.04% Coomassie), boiled for 5 min and loaded on the gel. TheHIS-tag of the expressed protein contains an X-press epitope thatenables detection of the HIS-CHIPS product by Western blot using theanti-X-press antibody (Invitrogen). In addition, CHIPS is specificallydetected with the polyclonal rabbit anti-CHIPS peptide antibody.Proteins are transferred to a nitrocellulose membrane, blocked with 4%gelatin in PBS and probed with the antibody and the appropriatesecondary peroxidase labeled conjugate (Harlow & Lane, 1988, Antibodies:a laboratory manual, Cold Spring Harbor Laboratory).

[0140] The concentration dependent inhibition of recombinant CHIPS(rCHIPS) on the expression of the receptor for fMLP (FPR) and C5a (C5aR)on neutrophils was demonstrated as follows. Cells were incubated withvarious concentrations rCHIPS for 15 min at room temperature, put on iceand subsequently probed with either BODIPY-labeled fMLP (see Example1.2) or a monoclonal antibody directed against the C5aR (clone 5 S/1,SeroTec) in combination with a secondary FITC-labeled goat-anti-mouse Ig(DAKO, 1:30). Finally cells were washed and analyzed for receptorexpression in a FACScan by measuring the fluorescence of 5000neutrophils. Receptor expression is compared to buffer treated cells andexpressed as a relative value.

[0141] The concentration dependent impairment of the intracellular freecalcium release induced by fMLP and C5a in neutrophils was tested asfollows. Cells were loaded with a Calcium specific intracellular probe(Fluo-3, acetoxymethyl (AM) ester; Molecular Probes) and incubated withvarious concentrations rCHIPS for 15 min at room temperature. From eachsample the initial fluorescence value was determined in the FACScan bymeasuring 2000 cells. Subsequently, stimulus was added (10⁻⁹ M fMLP or10⁻¹⁰ M rC5a) and the fluorescence intensity from the same sample wasdetermined exactly 15 seconds after administration of the stimulus (theoptimal time point for both agonists). Triggering neutrophils with fMLPor C5a initiates a rapid and transient increase in free intracellularCalcium concentration that is measured by an increase Fluo-3fluorescence signal. From each activated sample, the initial basalfluorescence value is subtracted. Results are expressed as a percentageof buffer treated cells stimulated with either fMLP or C5a.

Results

[0142]FIG. 11 is a representative image of an SDS-PAGE showing the finalpurified recombinant CHIPS (rCHIPS). The two flanking lanes (1 and 3)show the complete recombinant product that is encoded by the vectorgenerating the CHIPS protein with an additional Histidine tag andenterokinase cleavage site. This encodes for a protein with an apparentmolecular weight of 21 kDa, while purified Enterokinase treated CHIPSruns at an apparent molecular weight of 17 kDa, equally as shown fornatural purified CHIPS from S. aureus (see Example 1.1 and FIG. 2). Thepurified rCHIPS was characterized by MALDITOF MS and revealed amolecular mass of 14.12250, that is highly comparable with the predictedmolecular mass of 14.12217 based on the CHIPS sequence.

[0143]FIGS. 12 and 13 illustrate the biological effectiveness.

Example 7 Production of a Synthetic CHIPS Protein

[0144] It was demonstrated according to the invention that it ispossible to produce a synthetic polypeptide that has the exact sameactivity as natural and as recombinant CHIPS. The production process isas follows:

[0145] Synthesis ofFTFEPFPTNEEIESNKKMLEKEKAYKESFKNSGLPTTLGKLDERLRNYLKKGTKNSAQFEKMVILTENKGYYTVYLNTPLAEDRKNVELLGKMYKTYFFKKGESKSSYVINGPGKTNEYAYpeptide by TGT resin having 9-fluorenylmethyloxycarbonyl-and O t-butprotected Tyrosine [Fmoc Tyr (t-but)] attached thereto (5 g, 0.3 mmol,NovaBiochem) was transferred to peptide synthesiser, and a solution ofpiperidine (12 ml) in dimethylformamide (DMF; 18 ml) was added to theresin. The solution was swirled for 1 hour and the resin washed with DMF(3×30 ml) followed by dichloromethane (DCM; 3×30 ml) and allowed to dryunder vacuum for 5 minutes. The remainder of the amino acids weresequentially assembled employing standard Fmoc chemistry. Cleavage ofthe protein was accomplished by treating the protein resin with asolution of trifluoroacetic acid/tetraisopropylsilane/H₂O [90:8:2 v/v/v]for 2.5 hours. The crude product (2.1 gms) was isolated by etherprecipitation followed by purification by using High Performance LiquidChromatography. The purified product was characterised by MALDI MS.

[0146] References describing similar methods are: E Bayer et al., in:Peptides, Chemistry, Structure and Biology. Proceedings of the 13^(th)American Peptide symposium. R S Hodeges and J A Smith (eds) ESCOM,Leiden, (1994) p. 156.

[0147] G Grübler et al., in: Innovation and perspectives in Solid PhaseSynthesis 3^(rd) International Symposium. R E Pron (ed) MayflowerWorldwide, Birmingham (1994) p. 517.

Example 8 Competition for CHIPS Binding to its Putative ReceptorMaterial and Method

[0148] 8.1 Production of Recombinant CHIPS⁴⁻¹²¹

[0149] When several E. coli colonies containing the plasmid withrecombinant CHIPS were analyzed for proper insertion of the chp gene bysequencing, several incomplete insertions were found. One of them thatcontains the complete HIS-tag, enterokinase cleavage site and the CHIPSprotein minus the first three amino acids (CHIPS⁴⁻¹²¹; clone 19) wasfurther propagated and purified as described for complete CHIPS (seeExample 6).

[0150] 8.2 Competition With CHIPS-FITC Binding

[0151] In Falcon tubes 5 μl serial dilutions of recombinant CHIPS orCHIPS⁴⁻¹²¹ were prepared and mixed with 5 μl CHIPS-FITC (10 μg/ml; seeExample 2). Thereafter 50 μl isolated neutrophils at 5×10⁶ cells/ml areadded and incubated for 30 min on ice. Cells are washed and analyzed forCHIPS-FITC binding by flow cytometry as described in Example 2.

Results

[0152]FIG. 14 shows the concentration dependent inhibition of CHIPS-FITCbinding by both the complete recombinant CHIPS as well as therecombinant mutant CHIPS⁴⁻¹²¹. Both preparations show a similarinhibition pattern with equal effective concentrations.

1 5 1 24 DNA Artificial Sequence Primer sequence 1 gaaaaagaaa aagcatataaagaa 24 2 23 DNA Artificial Sequence chp-specific primer chp-5′ 2gaaaaagaaa ttagcaacaa cag 23 3 22 DNA Artificial Sequence chp-specificprimer chp-3′ 3 cataagatga tttagactct cc 22 4 602 DNA Staphylococcusaureus exon (125)..(488) 4 ataaatttaa atatagaatt taaggagaat taacatcattatgaaaaaga aattagcaac 60 aacagtttta gcattaagtt ttttaacggc aggaatcagtacacaccatc attcagcgaa 120 agct ttt act ttt gaa ccg ttt cct aca aat gaagaa ata gaa tca aat 169 Phe Thr Phe Glu Pro Phe Pro Thr Asn Glu Glu IleGlu Ser Asn 1 5 10 15 aag aaa atg tta gag aaa gaa aaa gct tat aaa gaatca ttt aaa aat 217 Lys Lys Met Leu Glu Lys Glu Lys Ala Tyr Lys Glu SerPhe Lys Asn 20 25 30 agt ggt ctt cct aca acg cta gga aaa tta gat gaa cgtttg aga aat 265 Ser Gly Leu Pro Thr Thr Leu Gly Lys Leu Asp Glu Arg LeuArg Asn 35 40 45 tat tta aag aaa ggc aca aaa aat tct gct caa ttt gaa aaaatg gtt 313 Tyr Leu Lys Lys Gly Thr Lys Asn Ser Ala Gln Phe Glu Lys MetVal 50 55 60 att tta act gaa aat aaa ggt tac tat aca gta tat ctg aat acacca 361 Ile Leu Thr Glu Asn Lys Gly Tyr Tyr Thr Val Tyr Leu Asn Thr Pro65 70 75 ctt gct gaa gat aga aaa aat gtt gag tta cta ggt aaa atg tat aaa409 Leu Ala Glu Asp Arg Lys Asn Val Glu Leu Leu Gly Lys Met Tyr Lys 8085 90 95 aca tac ttc ttt aaa aaa gga gag tct aaa tca tct tat gta att aat457 Thr Tyr Phe Phe Lys Lys Gly Glu Ser Lys Ser Ser Tyr Val Ile Asn 100105 110 ggt cct gga aaa act aat gaa tat gca tac t aatagtagtt acataaatta508 Gly Pro Gly Lys Thr Asn Glu Tyr Ala Tyr 115 120 aaaggtagatatttcttttt tatataaagg tttggcagac atttcataac ttgccaaacc 568 tttatatatctaattatcaa actgcactaa actt 602 5 121 PRT Staphylococcus aureus 5 Phe ThrPhe Glu Pro Phe Pro Thr Asn Glu Glu Ile Glu Ser Asn Lys 1 5 10 15 LysMet Leu Glu Lys Glu Lys Ala Tyr Lys Glu Ser Phe Lys Asn Ser 20 25 30 GlyLeu Pro Thr Thr Leu Gly Lys Leu Asp Glu Arg Leu Arg Asn Tyr 35 40 45 LeuLys Lys Gly Thr Lys Asn Ser Ala Gln Phe Glu Lys Met Val Ile 50 55 60 LeuThr Glu Asn Lys Gly Tyr Tyr Thr Val Tyr Leu Asn Thr Pro Leu 65 70 75 80Ala Glu Asp Arg Lys Asn Val Glu Leu Leu Gly Lys Met Tyr Lys Thr 85 90 95Tyr Phe Phe Lys Lys Gly Glu Ser Lys Ser Ser Tyr Val Ile Asn Gly 100 105110 Pro Gly Lys Thr Asn Glu Tyr Ala Tyr 115 120

1. An isolated nucleic acid molecule comprising a nucleotide sequenceencoding a (poly)peptide having CHIPS activity, said nucleotide sequencecorresponding to a sequence being selected from the group consisting of:a) a nucleotide sequence comprising at least part of the sequence asdepicted in FIG. 4 (SEQ ID NO 4); b) nucleotide sequences encoding a(poly)peptide having CHIPS activity and having the amino acid sequencedepicted in FIG. 5 (SEQ ID NO 5); c) nucleotide sequences encoding a(poly)peptide having CHIPS activity and having at least one portion ofthe amino acid sequence depicted in FIG. 5 (SEQ ID NO 5); d) nucleotidesequences being at least 40% identical to any one of the nucleotidesequences a), b) or c); e) nucleotide sequences hybridizing at stringentconditions with any one of the nucleotide sequences a), b), c) or d),and f) nucleotide sequences complementary to any of the nucleotidesequences a), b), c), d) or e).
 2. An isolated nucleic acid molecule asclaimed in claim 1, of which the part of the nucleotide sequence asdefined in claim 1 under a) corresponds to nucleotides 1 to 490 of FIG.4 (SEQ ID NO 4).
 3. An isolated nucleic acid molecule as claimed inclaim 1 or 2, of which the part of the nucleotide sequence as defined inclaim 1 under a) corresponds to nucleotides 41 to 490 of FIG. 4 (SEQ IDNO 4).
 4. An isolated nucleic acid molecule as claimed in claim 1, 2 or3, of which the part of the nucleotide sequence as defined in claim 1under a) corresponds to nucleotides 125 to 490 of FIG. 4 (SEQ ID NO 4).5. An isolated nucleic acid molecule as claimed in claims 1-4, whereinthe nucleotide sequence as defined in claim 1 under d) is at least 50%,preferably at least 60% , more preferably at least 70%, even morepreferably at least 80%, most preferably at least 90% identical to anyone of the nucleotide sequences a), b) or c).
 6. An isolated nucleicacid molecule as claimed in claims 1-5, wherein the stringent conditionsare constituted by overnight hybridisation at 42° C. in 5×SSC andwashing at 65° C. at 0.1×SSC.
 7. An isolated nucleic acid molecule asclaimed in claims 1-6, wherein the at least one portion of the aminoacid sequence as defined in claim 1 under c) constitutes alone or withother portions of the amino acid sequence the region(s) of the(poly)peptide having CHIPS activity that lead to biological activity. 8.An isolated nucleic acid molecule as claimed in claims 1-7, whichnucleic acid is DNA, RNA or cDNA.
 9. Recombinant vector comprising atleast one isolated nucleic acid molecule as claimed in claims 1-8. 10.Method for making a recombinant vector comprising inserting at least oneisolated nucleic acid molecule as claimed in claims 1-8 into a vector.11. Bacteriophage comprising at least one isolated nucleic acid moleculeas claimed in claims 1-8.
 12. Recombinant host comprising at least oneisolated nucleic acid molecule as claimed in claims 1-8, a vector asclaimed in claim 9 or a bacteriophage as claimed in claim
 11. 13.Recombinant host as claimed in claim 12, wherein the host is selectedfrom the group consisting of prokaryotes, protista, fungi, animals orplants.
 14. A recombinant host as claimed in claim 12 or 13, wherein thehost is selected from the group consisting of the bacteria Escherichiacoli, Bacillus subtilis, Staphylbcoccus aureus, the yeasts Saccharomycescerevisiae, Pichia pastoris, Candida, insect cells of the Drosophilasystem and the Baculovirus system, the mammalian cells monkey COS cells,hamster CHO, hamster BHK cells, human 293, human 3T3, human HeLa, humanU937, human Jurkat cells, mouse L cells.
 15. Method for producing arecombinant (poly)peptide having CHIPS activity, comprising culturing arecombinant host of claims 12-14 under conditions such that said(poly)peptide is expressed and recovering said (poly)peptide.
 16. Methodas claimed in claim 15, wherein the host is an Escherichia coli cell.17. Method as claimed in claim 15, wherein the host is a Staphylococcusaureus cell.
 18. Method as claimed in claim 17, wherein theStaphylococcus aureus cell is from a strain that already produces anendogenous protein having CHIPS activity (CHIPS).
 19. Method forproducing a synthetic (poly)peptide having CHIPS activity, comprisingdeducing the amino acid sequence encoded by a nucleic acid molecule asclaimed in claims 1-8 and synthetically producing a (poly)peptide havingthe said amino acid sequence.
 20. (Poly)peptide having CHIPS activityobtainable by any one of the methods as claimed in claims 15-19. 21.(Poly)peptide as claimed in claim 20 for use in diagnosis, prophylaxisor therapy.
 22. (Poly)peptide as claimed in claim 20 or 21 for use inthe treatment of acute and chronic inflammation reactions and HIVinfection.
 23. (Poly)peptide as claimed in claim 20 or 21 for use in thetreatment of Adult Respiratory Distress Syndrome (ARDS), ischaemicshock, traumatic brain injury, severe infections, myocardial infarction,stroke, vessel surgery, ulcerative colitis, Crohn's disease, ChronicObstructive Pulmonary Disease (COPD), rheumatoid arthritis, dermatoses,multiple sclerosis, Alzheimer's disease, arteriosclerosis, repetitivestrain injury (RSI), acute transplant rejection, burns, acute reactivearthritis, pancreatitis, vasculitis, glomerulonephritis, gout, frostbite and meningitis.
 24. Use of the (poly)peptide as claimed in claim 20for the manufacture of a therapeutic preparation for diagnosis,prophylaxis or therapy.
 25. Use as claimed in claim 24 for the treatmentof acute and chronic inflammation reactions and HIV infection.
 26. Useas claimed in claims 24 or 25 for the treatment of Adult RespiratoryDistress Syndrome (ARDS), ischaemic shock, traumatic brain injury,severe infections, myocardial infarction, stroke, vessel surgery,ulcerative colitis, Crohn's disease, Chronic Obstructive PulmonaryDisease (COPD), rheumatoid arthritis, dermatoses, multiple sclerosis,Alzheimer's disease, arteriosclerosis, repetitive strain injury (RSI),acute transplant rejection, burns, acute reactive arthritis,pancreatitis, vasculitis, glomerulonephritis, gout, frost bite andmeningitis.
 27. A therapeutic composition comprising a suitableexcipient and the (poly)peptide as claimed in claim
 20. 28. Acomposition as claimed in claim 27 for treating acute and chronicinflammation reactions and HIV infection.
 29. A composition as claimedin claim 27 for treating Adult Respiratory Distress Syndrome (ARDS),ischaemic shock, severe infections, myocardial infarction, stroke,vessel surgery, ulcerative colitis, Crohn's disease, Chronic ObstructivePulmonary Disease (COPD), rheumatoid arthritis, dermatoses, multiplesclerosis, Alzheimer's disease, arteriosclerosis, repetitive straininjury (RSI), acute transplant rejection, burns, acute reactivearthritis, pancreatitis, vasculitis, glomerulonephritis, gout, frostbite and meningitis.
 30. An antibody or biologically active fragmentthereof specifically directed to the (poly)peptide as claimed in claim20.
 31. An antibody or biologically active fragment thereof as claimedin claim 30 for use in diagnosis, prophylaxis or therapy.
 32. Anantibody or biologically active fragment thereof as claimed in claim 30or 31 for use in the treatment of staphylococcus infection.
 33. Use ofan antibody as claimed in claim 30 for the manufacture of a therapeuticpreparation for diagnosis, prophylaxis or therapy.
 34. CHIPS-based,CHIPS-receptor blocking molecule characterised by its ability to competewith CHIPS in a CHIPS binding assay as described in Example
 8. 35.CHIPS-based, CHIPS-receptor blocking molecules as claimed in claim 34for use in diagnosis, prophylaxis or therapy.
 36. CHIPS-based,CHIPS-receptor blocking molecules as claimed in claim 34 or 35 for usein the treatment of staphylococcus infection.
 37. Use of CHIPS-based,CHIPS-receptor blocking molecules as claimed in claim 34 for themanufacture of a therapeutic preparation for diagnosis, prophylaxis ortherapy.
 38. Use as claimed in claim 33 or 37 for the treatment ofstaphylococcus infection.
 39. Therapeutic composition comprising asuitable excipient and one or more antibodies as claimed in claim 30and/or biologically active fragments thereof and/or one or moreCHIPS-based, CHIPS-receptor blocking molecules as claimed in claim 34.40. An isolated nucleic acid molecule for use in gene therapy. 41.Method for treating a subject suffering from inflammation and AIDS byadministering a therapeutically effective amount of a (poly)peptide asclaimed in claim
 20. 42. Method for gene therapeutically treating asubject suffering from inflammation and AIDS by administering atherapeutically effective amount of a nucleic acid molecule as claimedin claims 1-8.
 43. Method for treating a subject suffering fromstaphylococcus infection by administering a therapeutically effectiveamount of an antibody and/or biologically active fragment thereof asclaimed in claim 30 and/or one or more CHIPS-based, CHIPS-receptorblocking molecules as claimed in claim
 34. 44. Method for isolating froman organism a gene encoding a protein having CHIPS activity, comprisingscreening of a genomic or cDNA library of that organism with a probebased on the nucleic acid molecule as claimed in claims 1-8, isolationof the positive clones, and testing whether the positive clones showCHIPS activity.
 45. Method for identifying nucleic acid sequencesencoding a (poly)peptide having CHIPS activity, comprising comparison ofthe sequence as depicted in FIG. 4 (SEQ ID NO 4) with the nucleic acidsequence information contained in a database.
 46. Method for identifyingamino acid sequences of a (poly)peptide having CHIPS activity,comprising comparison of the sequence as depicted in FIG. 5 (SEQ ID NO5) with the nucleic acid sequence information contained in a database.47. Micro-organism harboring a nucleic acid molecule as claimed inclaims 1-8 for use as a medicament for the treatment of acute andchronic inflammation reactions and HIV infection.
 48. Micro-organism asclaimed in claim 47 for treating Adult Respiratory Distress Syndrome(ARDS), ischaemic shock, severe infections, myocardial infarction,stroke, vessel surgery, ulcerative colitis, Crohn's disease, ChronicObstructive Pulmonary Disease (COPD), rheumatoid arthritis, dermatoses,multiple sclerosis, Alzheimer's disease, arteriosclerosis, repetitivestrain injury (RSI), acute transplant rejection, burns, acute reactivearthritis, pancreatitis, vasculitis, glomerulonephritis, gout, frostbite and meningitis.
 49. Method for producing (poly)peptide(s) havingCHIPS activity, comprising culturing wildtype, non-recombinant,Staphylococcus strains that produce endogenous chemotaxis inhibitory(poly)peptide(s) and recovering same.
 50. (Poly)peptide having an aminoacid sequence that is at least 40% homologous to the amino acid sequencedepicted in FIG. 5 (SEQ ID NO 5) and having CHIPS activity. 51.(Poly)peptides having an amino acid sequence that is identical to one ormore portions of the amino acid sequence depicted in FIG. 5 (SEQ ID NO5) and having CHIPS activity.
 52. (Poly)peptides as claimed in claims 50and 51 for use in the identification of competitors for CHIPS binding.53. Diagnostic test for use in diagnosing patients infected withStaphylococcus aureus for the presence in the infecting S. aureus of theCHIPS gene, which test is a PCR test for which the primers are designedon the basis of the nucleotide sequence depicted in FIG. 4 (SEQ ID NO4).